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NMR tools for drug design validated on phosphatases

Final Report Summary - NDDP (NMR Tools for Drug Design Validated on Phosphatases)

The instruments for drug discovery include high-throughput synthesis and subsequent screening, biological assays, molecular biology, computational modelling, electron microscopy, X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy. Only NMR and X-ray diffraction provide high-resolution information about the protein-ligand interactions at atomic resolution. While the technology for high-throughput X-ray crystallography of protein-ligand complexes has been optimised such that it is now routinely used in all major pharmaceutical companies, this is not the case for NMR spectroscopy, despite its tremendous potential, relative to and complementary to X-ray crystallography. NMR can provide both structural and dynamic information on the protein-ligand complex at atomic resolution, information that is highly desirable for efficient drug design. Potentially, NMR can provide such information very rapidly and without the constraints of co-crystallisation.

In this project we developed a set of NMR tools for targeted drug design, including smart ligand screening, accurate determination of protein ligand interaction and fast structure determination that can overcome the current barriers for the routine use of NMR for efficient drug design. In our protocol X-ray structures are used as molecular models that provide a very detailed picture of the protein. Knowing the X-ray structure of the protein target, our project provides a protocol for the rapid identification of protein-ligand complexes using modern NMR techniques. This can significantly speed up drug design efforts for typical drug targets and shorten the lead-time for new drugs. The NMR parameters allow a structural and dynamic characterisation of drug-receptor interactions at atomic resolution and can be used as input for new, fast computer modelling approaches for protein-inhibitor complexes. The protocol combines the considerable strengths of NMR for screening diverse inhibitors and solution conditions with power of X-ray crystallography for structure determination exploiting optimally the complementary strengths of both techniques.

In this project, phosphatases were selected as the class of proteins, which were used to develop a strategy for NMR based drug design. The typical molecular weight of phosphatases is below 50 kDa and therefore amenable to NMR-studies using high-field instrumentation. Phosphatases are a major class of drug targets for a broad range of medical indications. The majority of cellular functions depend on phosphorylation by kinases and dephosphorylation by phosphatases. High eukaryotes encode approximately 500 protein kinase and 100 protein phosphatase schemes, corresponding to 3 % of their genome. While the importance of kinases in cellular regulation has led to substantial drug design activities, the importance of phosphatases has only been appreciated recently. Protein phosphatases regulate insulin signalling, cell growth and the cell cycle. Therefore, the inhibition of phosphatases is perused for the treatment of diabetes and obesity and various types of cancer. The availability of the human genome provides access to a broad variety of phosphatases and allows systematic drug design using sophisticated techniques to identify potential inhibitors.

Thus our project resulted in the NMR characterizations of phosphatase complexes and in protocol that integrates X-ray crystallography, NMR screening and computational modelling for fast and efficient drug. It turned out that the protein production of phosphatases was extremely difficult, but the protocol could successfully validated on several other protein-ligand complexes, among which a protein kinase A complex and the phosphatase PTP1b. A series of cycloocta[b]indoles were synthesised and investigated for their inhibitory potential in enzymatic-activity assays with the tyrosine phosphatase PTP1B from Mycobacterium tuberculosis. The screen yielded potent inhibitors of the tyrosine phosphatase MptpB that may be a promising scaffold for the development of novel antibiotic agents with activity against Mycobacterium tuberculosis.